Ad.TERT-TRAIL对肿瘤细胞的杀伤作用及其机理

背景与目的：有学者用人端粒酶逆转录酶(TERT)启动子替换野生型腺病毒E1A自身的启动子,构建了一种肿瘤特异性增殖病毒,即溶瘤腺病毒Ad．TERT。细胞实验和动物实验表明,Ad．TERT有很好的抗癌作用。本研究目的是要在Ad．TERT中插入凋亡基因trail生成Ad．TERT—TRAIL,观察其是否具有更强的杀伤肿瘤细胞的功效并研究其机理。方法：通过腺病毒包装质粒pBHGE3与携带trail的小质粒pZhTERT—trail在HEK293细胞中同源重组构建Ad．TERT-TRAIL病毒．然后用PCR鉴定。并用Western blot检测E1A、trail基因的表达以进一步确证。用结晶紫染色法和MTT法检测Ad．TERT和Ad．TERT—TRAIL对肿瘤细胞和正常细胞杀伤作用的差异。用Western blot检测Caspase-3表达量以观察肿瘤细胞的凋亡情况,同时用流式细胞仪测定肿瘤细胞的凋亡率。结果：PCR扩增出来的条带为860bp．即为trail基因。Western blot结果显示．Ad．TERT—TRAIL中的E1A和trail只在肿瘤细胞中表达,说明Ad．TERT—TRAIL构建成功。结晶紫染色结果表明,Ad．TERT-TRAIL对肿瘤细胞的杀伤力比Ad．TERT高10～100倍．而对正常细胞的影响跟Ad．TERT一样小。100MOI(multiple of infection)Ad．TERT—TRAIL感染结肠癌细胞SW620 72h后．细胞存活率为4％：而感染了Ad．TERT的SW620细胞存活率却达56％。两种病毒对正常细胞NHLF的影响都比较弱,存活率在85％以上。随着作用时间的延长．感染了Ad．TERT—TRAIL和Ad．TERT的SW620细胞中Caspase-3的表达量都在升高,但前者的表达量明显大于后者．说明前者所引起的肿瘤细胞的凋亡程度比后者高。流式细胞仪检测结果显示,Ad．TERT—TRAIL引起SW620细胞的凋亡率是Ad．TFRT的4倍。结论：携带基因的溶瘤腺病毒Ad．TERT—TRAIL比单纯的溶瘤病毒Ad．TERT对肿瘤细胞的杀伤力更强,这种作用可能与trail基因能诱导Caspase-3表达量增加、大量肿瘤细胞凋亡有关。     

癌特异性双靶向载体的安全性及其携带基因的杀伤性

背景与目的:我们创导的“癌症的靶向双基因-病毒治疗”策略可消灭移植性肿瘤,目前最重要的是要提高其安全性,使其只靶向肿瘤细胞,而在正常细胞内不复制或者很少复制。本研究旨在构建肿瘤特异性的双靶向腺病毒TD55,并研究其安全性;在TD55中插入凋亡基因TRAIL,构建成TD55-TRAIL,研究其杀伤性。方法:用hTERT启动子控制腺病毒复制必需的E1A基因,并将E1B55KD基因删除,构建肿瘤特异性的双靶向腺病毒TD55,使其只靶向p53功能缺乏的肿瘤。在TD55中插入凋亡基因TRAIL,构建成TD55-TRAIL,通过分子克隆构建质粒pTD55和pTD55-TRAIL,并与包装腺病毒的大质粒pBHGE3在293细胞中同源重组,得到腺病毒TD55和TD55-TRAIL。构建好的病毒体外感染人胚肺细胞MRC5、WI38,人结肠癌细胞SW620、HCT116、人肺癌细胞A549,分别用结晶紫染色法和MTT法检测其对细胞生长的影响。用流式细胞仪测定肿瘤细胞的凋亡率。结果:结晶紫染色结果和MTT结果表明,双靶向腺病毒TD55-TRAIL对正常细胞MRC5和WI38的杀伤作用比ZD55-TRAIL小很多。病毒复制能力分析表明,单靶向腺病毒在正常细胞中的复制倍数是双靶向腺病毒的3～5倍。TD55和TD55-TRAIL均能引起SW620细胞的凋亡,后者是前者的3.3倍。结论:双靶向腺病毒TD55-TRAIL对正常细胞的安全性比以往所构建的单靶向腺病毒ZD55-TRAIL更好,说明双靶向载体TD55比单靶向载体ZD55靶向性更高,如做为治疗药物可增加安全性,在治疗中可大大增加药物的安全性。携带的TRAIL基因能引起肿瘤细胞的凋亡,杀伤力较TD55增加数倍。     

XIAP siRNA对TRAIL诱导结肠癌细胞SW620凋亡能力的影响

 目的
研究XIAP siRNA对肿瘤坏死因子相关的凋亡诱导配体(Tumor Necrosis Factor-related Apoptosis-Inducing Ligand,TRAIL)诱导结
肠癌细胞SW620凋亡的影响。方法SW620细胞分为转染XIAP siRNA组、空转染对照组或者siRNA阴性对照组,然后给予TRAIL处理。XIAP
表达水平的变化、细胞增殖抑制、Caspase-3活性分别由Western blot、MTT法和Caspase-3荧光测定来检测。切割PARP的表达水平由
Western blot来测定。结果XIAP siRNA转染以后显著下调XIAP蛋白表达水平。和空转染对照组、siRNA阴性对照组相比,XIAP siRNA
显著增强10、100、1 000 ng/ml等浓度的TRAIL作用以后SW620细胞的增殖抑制、Caspase-3活性和激活PARP。结论 XIAP siRNA能显
著增强TRAIL诱导结肠癌细胞SW620的凋亡。     

TRAIL与阿霉素联用协同杀伤人结肠癌细胞SW480

背景与目的:肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactor-relatedapoptosisinducingligand,TRAIL)可选择性杀伤肿瘤细胞,而不影响正常细胞生长。当一部分肿瘤细胞对TRAIL不敏感时,特定的其它药物可增强其杀伤作用。本文旨在探讨结肠癌细胞SW480对TRAIL的敏感性,以及TRAIL与阿霉素联用对细胞的杀伤作用及可能作用机制。方法:常规培养结肠癌细胞SW480。利用MTT法检测细胞毒性作用,流式细胞术定量分析凋亡细胞比例,透射电镜在亚细胞结构形态上证实凋亡细胞,Westernblot分析p53及bcl-2蛋白表达变化。结果:(1)SW480细胞对TRAIL不敏感,100ng/mlTRAIL只能杀伤7.8%的细胞,IC50>1000ng/ml,且不存在浓度依赖性。(2)SW480细胞对阿霉素敏感,存在浓度依赖性作用,IC50=65μmol/L,0.86μmol/L的阿霉素对细胞不表现杀伤作用。(3)TRAIL与阿霉素合用表现出协同作用,亚毒性浓度TRAIL(100ng/ml)与亚毒性浓度阿霉素(0.86μmol/L)联用可杀伤80%SW480细胞。流式细胞学证实这种杀伤作用主要通过诱导细胞凋亡实现,透射电镜亦观察到大量凋亡细胞存在。药物作用前后,p53及bcl-2蛋白表达水平无明显改变。结论:结肠癌细胞株SW480对TRAIL不敏感,但TRAIL与亚毒性浓度阿霉素联用对癌细胞有协同杀伤作用,这种细胞毒性作用主要表现     

hTERT基因启动子调控HSV-tk/GCV系统对HepG2细胞杀伤作用的实验研究

目的:研究人端粒酶逆转录酶(hTERT)基因核心启动子调控的人单纯疱疹病毒胸苷激酶/更昔洛韦(HSV-tk/GCV)系统对肝癌细胞体外杀伤作用.方法:以pGL3-hT-ERT/tk为基础,构建pGL3-hTERT/lue、pGL3-basie/tk、pGL3-control/tk等真核表达载体,分别将其用脂质体法转染HepG2肝癌细胞和正常肝细胞L02,荧光显微镜观察转染效果,给予前药GCV,用原位末端标记(TUNEL)法、流式细胞仪检测hTERT调控的HSV-tk/GCV系统对两种细胞的杀伤作用,Western blot检测细胞周期素Cyclin D1、CDK2、p21蛋白表达.结果:hTERT启动子调控的HSV-tk/GCV系统对肝癌细胞有明显的杀伤作用,使41.85%的细胞凋亡;而对正常细胞作用不明显,Western也显示Cyclin D1、CDK2蛋白表达下降而p21升高.结论:hTERT启动子调控的tk基因治疗是一种具有肿瘤特异性的治疗方法,有望解决肿瘤基因治疗中的特异性杀伤及降低毒副反应等问题.     

含甲胎蛋白肽段的重组腺病毒转染树突状细胞诱导细胞毒性T细胞对肝癌细胞的体外杀伤效应

目的 研究人外周血单核细胞来源的树突状细胞(DC)转染含甲胎蛋白(AFP,137-145)片段的重组腺相关病毒后所诱导的特异性T细胞对肝癌细胞株HepG2和SMMC-7721的体外杀伤作用.方法 抽取健康志愿者外周血,分离单核细胞,体外培养,使用含AFP片断的重组腺相关病毒转染未成熟DC,诱导特异性T细胞.检测体外培养的DC和细胞毒性T细胞(CTL)活性,应用四甲基偶氮唑蓝(MTT)法检测CTL对HepG2和SMMC-7721细胞的杀伤作用.结果 转染或未转染的体外培养的成熟DC高表达CD40、CD86和IL12,成熟DC诱导的CTL高表达IFNγ;修饰成熟后的DC后体外能诱导特异性CTL,该CTL在休外对肝癌细胞株HepG2和SMMC-7721均有杀伤作用.结论 重组腺相关病毒转染DC,不明显改变DC表型和刺激淋巴细胞增殖和分化功能,可诱导自体CTL增殖,含AFP(137～145)片断的腺相关病毒转染DC诱导自体CTL对肝癌细胞株HepG2和SMMC-7721细胞有明显杀伤作用,DC疫苗可以作为肝癌患者免疫治疗的有效补充.     

肿瘤细胞对TRAIL敏感性与其表面DR5表达水平的相关性研究

目的　探讨肿瘤细胞表面DR5表达水平与其对肿瘤坏死因子相关的凋亡诱导配体(TRAIL)敏感性之间的关系。方法　利用抗DR5特异性单克隆抗体 ,采用流式细胞仪技术直接检测不同肿瘤细胞系表面DR5的表达水平 ,并采用TRAIL凋亡检测试剂盒检测肿瘤细胞对TRAIL诱导凋亡的敏感性 ,研究两者之间的关系。结果　不同肿瘤细胞表面DR5的表达水平分别为 :U937细胞97.9%、Jurkat细胞 95 .1%、SW4 80细胞 93.8%、HCT116细胞 86 .2 %、HL 6 0细胞 6 4 .2 %、HeLa细胞4 6 .6 %、K5 6 2细胞 13.1% ;TRAIL诱导的细胞凋亡率分别为 :U937细胞 72 .6 %、Jurkat细胞 85 .2 %、SW4 80细胞 78.6 %、HCT116细胞 70 .2 %、HL 6 0细胞 6 0 .1%、HeLa细胞 4 5 .4 %、K5 6 2细胞 12 .3%。经统计学分析 ,两者之间呈现非常明显的正相关 (r=0 .997,P <0 .0 0 1)。结论　肿瘤细胞对TRAIL的敏感性与其表面DR5表达水平有关 ,表明DR5的表达水平在TRAIL诱导细胞凋亡方面起着十分重要的作用     

负载hTERT基因片段的DC肿瘤疫苗制备及其抗肿瘤作用的研究

目的:研究负载hTERT基因片段的树突状细胞(DC)肿瘤疫苗及其抗肿瘤作用.方法:构建负载hTERT基因片段的慢病毒载体,采用细胞免疫化学技术检测Lenti-hTERT转导的293T细胞中hTERT基因的表达;分离制备脐血来源的DC,通过相差显微镜和流式细胞仪鉴定; Lenti-hTERT转导DC细胞制备DC疫苗; DC疫苗刺激同种T淋巴细胞诱导特异性CTL并用MTT法检测其增殖能力和对靶细胞的特异性杀伤能力.结果:成功构建负载hTERT基因片段的Lenti-hTERT,病毒滴度达5.88×106 U/mL;转导后细胞中hTERT的表达显著升高,持续时间＞2个月; 分离制备脐血来源的DC,经慢病毒转导成为负载hTERT的DC疫苗,负载了hTERT抗原的DC刺激淋巴细胞增殖的能力明显优于未负载hTERT的DC(P＜0.01),增殖指数均>1.5.负载了hTERT抗原的DC诱导CTLT对HepG2肝癌细胞的杀伤能力明显高于未经修饰的DC所诱导CTLN(P＜0.01),抑瘤率分别为54.78%和12.08%;对hTERT阴性的293T细胞CTLN和CTLT的抑制率均较低且差异无统计学意义,P>0.05.结论:用慢病毒介导hTERT修饰制备的DC疫苗,其刺激T细胞增殖的能力增强,诱导的CTL对端粒酶阳性的HepG2肿瘤细胞具有特异而且显著增强的杀伤效应.     

重组人可溶性凋亡素-2在人喉癌株中表达及其体外诱导凋亡作用的实验研究

目的:研究凋亡因子TRAIL结合端粒酶启动子对肿瘤的特异性杀伤作用。方法:刺激人外周血淋巴细胞增殖,提取总RNA。采用RTPCR扩增IL2信号肽基因和TRAIL基因的融合基因,并克隆入真核表达载体pGL3181hTERT肿瘤特异性端粒酶启动子的下游,构建TRAIL基因的重组真核表达载体pGL3181hTERT/TRAIL。用Westernblot鉴定喉癌细胞株Hepa2中表达产物。将该重组载体经阳离子脂质体转染入人喉癌细胞株Hepa2中,用电镜观察细胞的形态变化,用流式细胞仪技术(FCM)分析转染后细胞凋亡率及细胞周期的变化。结果:PCR扩增得到了613bp的cDNA片断。与GenBank中报道的IL2信号肽和TRAIL凋亡诱导功能区cDNA序列完全一致,成功构建了重组真核表达载体pGL3181hTERT/TRAIL。Westernblot结果显示,获得的瞬时转染TRAIL在喉癌细胞Hepa2中特异表达,且能诱导其凋亡。结论:重组真核表达载体pGL3181hTERT/TRAIL的成功构建,为肿瘤基因治疗的靶向性提供了可行的理想工具。     

TRAIL与羟基喜树碱联用协同杀伤人肺腺癌A549细胞

目的：探讨羟基喜树碱与肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis inducing ligand，TRAIL）联用对肺腺癌A549细胞是否具有协同杀伤作用，并对其机制进行初步研究。方法：MTT法检测细胞毒性作用，流式细胞仪检测细胞凋亡和线粒体膜电位，Western blot分析bcl-2和bcl-xl蛋白水平变化。结果：单用100ng／mL TRAIL，4、40和400μg／mL羟基喜树碱对A549细胞杀伤率分别为13．9％、3．0％、23．4％和76．7％，联合用药的抑制率分别为43．6％、52．5％和83．1%。100ng／mL TRAIL和4μg/mL羟基喜树碱有协同作用，可使细胞线粒体膜电住降低，但不影响bcl-2和bcl-xl蛋白表达。结论：TRAIL与羟基喜树碱联用对肺腺癌A549细胞有明显的协同杀伤作用，其机制可能与羟基喜树碱降低A549细胞线粒体膜电位有关。     

A mitochondrial block and expression of XIAP lead to resistance to TRAIL-induced apoptosis during progression to metastasis of a colon carcinoma

A pair of isogenic colon carcinoma cells, SW480 and 620, was used to investigate the mechanisms of acquired tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance during tumour progression. Whereas primary tumour SW480 cells are sensitive to TRAIL-induced apoptosis, metastatic SW620 cells are resistant. The apoptotic signalling activated by TRAIL in SW480 cells is a type II pathway. We show that in SW620 cells, although caspase-8 is recruited and activated at the death-inducing-signalling complex and Bid is cleaved, this does not lead to caspase-9 activation. Comparison of Bcl-2, Bcl-xL and Mcl-1 levels in both cell lines showed no difference. In SW620 cells transfected with a tBid-GFP construct, tBid-GFP was correctly localized to the mitochondria. Thus, the resistance of SW620 cells is at the level of the mitochondria that can withstand large amounts of tBid. Although caspase-3 was directly cleaved by caspase-8 in SW620 cells to yield the p20 fragment, no further autocatalytic maturation into the p17 fragment was observed. We show that, in contrast to SW480 cells, the SW620 cell line expresses high amounts of X-linked inhibitor of apoptosis (XIAP). Downregulation of XIAP with bortezomib or small-interfering RNA was sufficient to restore the sensitivity of SW620 cells to TRAIL-induced apoptosis in the absence of SMAC/Diablo or cytochrome c release from the mitochondria. Thus, SW620 cells have developed a dual resistance to TRAIL-induced apoptosis: a block at the level of the mitochondria and, after a conversion to a type I pathway, an increased expression of XIAP which inhibits this pathway.     

EGFR inhibitors sensitize non-small cell lung cancer cells to TRAIL-induced apoptosis

Apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be regulated by the epidermal growth factor (EGF) signaling pathway. In this study, recombinant adenoviral vectors that encode TRAIL gene from the hTERT/RGD promoter (AdTRAIL) was combined with drugs including gefitinib, elotinib, and cetuximab that inhibit EGFR and the EGF signaling pathway in non-small cell lung cancer (NSCLC) cell lines to investigate their antitumor activity. In vitro, compared to single reagent, AdTRAIL combined with EGFR inhibitors reduced proliferation and enhanced apoptosis in H460, A549, and SW1573 cell lines. Western blot results suggested that these effects were relative to up-regulation of pro-apoptosis protein BAX and down-regulation of p-AKT. In vivo, AdTRAIL combined with cetuximab resulted in a significant growth reduction in H460 xenografts without damage to the main organs of nude mice. Histological examination and TUNEL analyses of xenografts showed that cetuximab enhanced cell apoptosis induced by AdTRAIL. These results indicate that EGFR inhibitors enhanced AdTRAIL anti-tumor activity in NSCLC cell lines and that inhibiting the AKT pathway played an important role in this enhancement.     

Oridonin-induced apoptosis in SW620 human colorectal adenocarcinoma cells

Oridonin, a diterpenoid isolated from Rabdosia rubescens (Hemsl.) Hara, inhibited the growth of human tumor cell lines SW620 (colon), MCF-7 (breast) and K562 (bone marrow), and induced significant levels of apoptosis in SW620. Morphological changes indicative of cell apoptosis were observed after the cells were exposed to oridonin for 24 h. Growth inhibition was associated with G1 phase arrest, and with time- and dose-dependent increases in caspase-3 activity. We therefore conclude that oridonin inhibits the proliferation of SW620 cells by induction of apoptosis via the activation of caspase-3. Our data suggest that oridonin may have significant potential as an anti-colorectal adenocarcinoma agent.     

Long-term tumor-free survival from treatment with the GFP-TRAIL fusion gene expressed from the hTERT promoter in breast cancer cells

We evaluated anti-tumor activity and toxic effect of an adenoviral vector expressing the GFP/TRAIL fusion gene from the hTERT promoter (designated Ad/gTRAIL) on human breast cancer cell lines and on normal human breast cells. Treatment with Ad/gTRAIL elicited high levels of transgene expression and apoptosis in a variety of breast cancer cell lines. Furthermore, treatment with Ad/gTRAIL was effective in killing breast cancer lines resistant to doxorubicin or soluble TRAIL protein. In contrast, only minimal transgene expression and toxicity was detected in normal human primary mammary epithelial cells after treatment with this vector. An in vivo study further showed that the intralesional administration of Ad/gTRAIL effectively suppressed the growth of human tumor xenografts derived from both doxorubicin-sensitive and doxorubicin-resistant breast cancer lines. Specifically, about 50% of animals bearing doxorubicin-sensitive and doxorubicin-resistant breast cancer xenografts showed complete tumor regression and remained tumor-free for over 5 months. These results suggest that the adenovirus encoding the GFP/TRAIL gene driven by the hTERT promoter has potential application in cancer therapy.     

Potent antitumor effect elicited by RGD-mda-7, an mda-7/IL-24 mutant, via targeting the integrin receptor of tumor cells

The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, an mda-7/IL-24 mutant, RGD-mda-7, which includes the cell adhesive sequence 164Arg-165Gly-166Asp (RGD motif), was constructed and evaluated for bioactivity. RGD peptide binds to integrins α(V)β(3) and α(V)β(5), which are selectively expressed in tumor neovasculature and in the surface of some tumor cells. The wtmda-7/IL-24 and RGD-mda-7 were expressed in Escherichia coli and then purified and renatured. The immunostimulatory activity of RGD-mda-7 was assayed by stimulating peripheral blood mononuclear cells. The results suggested that the abilities of RGD-mda-7 to induce IL-6, TNF-α, and IFN-γ production were higher than wtmda-7/IL-24. Tumor targeting of RGD-mda-7 was assayed using cell adhesion experiments. The antitumor effect of the purified RGD-mda-7 on cell proliferation in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake, cell apoptosis by staining with fluorescent probes of FITC-annexin V and DAPI, and caspase-3 expression and activity. The in vitro results showed that RGD-mda-7 inhibited the proliferation of multiple tumor cell lines (Hela, ACHN, HepG2, and A549). Staining with fluorescent probes of FITC-annexin V and DAPI indicated that RGD-mda-7 could induce apoptosis more effectively in four tumor cell lines than wtmda-7/IL-24, but has no effect on normal cell line NHLF. Western blotting showed that treatment of tumor cells with RGD-mda-7 could activate apoptotic pathway by cleavage of caspase-3 as same as wtmda-7/IL-24. Further, RGD-mda-7 group showed a higher cleaved level of caspase-3, but not in NHLF cells. These results demonstrate that RGD-MDA-7 possesses more potent antitumor effects than wtmda-7/IL-24 and therefore merits further investigation in preclinical and clinical studies.     

Effects of TNF Secreting HEK Cells on B Lymphocytes’ Apoptosis in Human Chronic Lymphocytic Leukemias

Background: Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) is an antitumorcandidate in cancer therapy. This study focused on effects of TRAIL, as a proapototic ligand that causes apoptosis,in B-CELL chronic lymphocytic leukemia cells (B-CLL) . Materials and Methods: A population of HEK 293cells was transducted by lentivirus that these achieved ability for producing the TRAIL protein and then HEK293 cells transducted were placed in the vicinity of CLL cells. After 24 hours of co-culture, apoptosis of CLLcells was assessed by annexin V staining. Results: The amount of Apoptosis was examined separately in fourgroups: 293 HEK TRAIL (16.17±1.04%); 293 HEK GFP (2.7±0.57%); WT 293 HEK (2±2.6%); and CLL cells(0.01±0.01%). Among the groups studied, the maximum amount of apoptosis was in the group that the vectorencoding TRAIL was transducted. In this group, the mean level of soluble TRAIL in the culture medium was253pg/ml; also flow cytometry analyzes showed that proapotosis in this group was 32.8±1.6%, which was higherthan the other groups. Conclusions: In this study, we have demonstrated that TNF secreted from HEK 293 cellsare effective in death of CLL cells.